With its ability to secrete high levels of recombinant proteins, the yeast Pichia pastoris is extensively used for protein manufacturing. However, as actual yields vary widely for individual proteins, GlycoFi is focusing on the improvement of protein yields in P. pastoris as part of the company's long term mission to develop yeast-based protein production technology for manufacturing therapeutic proteins with human glycosylation structures. In Phase I of this project a strategy was developed and tested using the model protein alpha 1-antitrypsin for overcoming bottlenecks in yeast secretion that result in less than optimal yields. A combinatorial library/high throughput screening methodology was designed to optimize translocation of the nascent peptide into the endoplasmic reticulum (ER) and protein folding within the ER. In Phase II, yield optimization studies will include IgG as well as alpha 1-antitrypsin with specific experiments targeting expression and secretion of the IgG H and L chains. The toolkit created in Phase I will be expanded through the implementation of several new high throughput approaches toward yield improvement including mutagenesis and cDNA library screening and deletion of protease genes as well as traditional fermentation and purification process development. Proposed Commercial Application: GlycoFi's Humanized Yeast(tm) enables the study of glycosylation structure-function relationships and creates the ability not only to pinpoint the specific glycoforms that optimize the bioactivity, pharmacokinetics and overall efficacy of a therapeutic protein, but also to manufacture them at low cost with a high degree of uniformity. GlycoFi's technology will provide the biopharmaceutical industry with a high capacity alternative to mammalian cell culture that will allow life-improving drugs to reach the clinic in a faster, more cost-effective manner.